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CHROMOSOME BANDING TECHNIQUES

INTRODUCTION:A chromosome banding pattern is comprised of alternating light and dark stripes, or bands that appear along its length after being stained with a dye. OR The treatment of chromosomes to reveal characteristic patterns of horizontal bands like bar codes is known as chromosomal banding A unique banding pattern is used to identify each chromosome and to diagnose chromosomal aberrations, including chromosome breakage, loss, duplication or inverted segments.

Most of chromosomes, at the prophase and the metaphase, are characterized by a banding pattern. But this banding pattern is more evident and clear in the case of larger chromosomes such as the polytene chromosomes of drosophila melanogaster, or the fruit fly.

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The banding patterns are the regions rich in heterochromatin, where the histone -DNA interaction is more. These complexes can be stained very easily by the conventional nuclear dyes or chromosomal dyes such as orcein.

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The regions between the bands are actually the active regions of the chromatin where more genes are present, but the quantity of DNA is very low and therefore the histone proteins. That is why they appear unstained or colored lightly. These interband regions can be detected with immuno-staining by using fluorescently labeled antibodies against the DNA-dependent RNA polymerase, which is usually seen with euchromatin regions required for the process of transcription.

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Specialized staining techniques are now available, which enable one to differentiate or precisely identify individual chromosome homologes, chromosome regions, and/ or chromosome bands. A renewed interest in the chromosomal or cytogenetic status of various species has been generated by the advancements of genetic mapping techniques utilizing fluorescence in situ hybridization or FISH.

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Depending upon the type of dye or fluorochrome or the chromosome pretreatment, there can be different types of banding patterns. They include banding patterns such as G-banding, Q-banding, C-banding, and R-banding etc.

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The data generated by multiple chromosome banding techniques can be used for karyotypic analysis. At a conference in Paris in 1971 , the bands visualized in human mid- metaphase karyotypes by such techniques were assigned a nomenclature in which letters p & q represent respectively, the short & long “arms” of a metaphase chromatid ; these arms are then subdivided by numbers.

3 • ISCN is International System For Human Cytogenetic Nomenclature. Each area of chromosome is given number, lowest number closest (proximal) to centromere &

DIFFERENT CHROMOSOME BANDING TECHNIQUES
1. Q-banding: • Quinacrine mustard, an alkylating agent, was the first chemical to be used for chromosome banding. T. Caspersson and his colleagues, who developed the technique, microscope. • Quinacrine dihydrochloride was subsequently substituted for quinacrine mustard. • The alternating bands of bright and dull fluorescence were called Q bands. Quinacrinebright bands were composed primarily of DNA that was rich in the bases adenine (A) and thymine (T), and quinacrine-dull bands were composed of DNA that was rich in the bases guanine (G) and cytosine (C). noticed that bright and dull fluorescent bands appeared after chromosomes stained with quinacrine mustard were viewed under a fluorescence

4 • This banding pattern is obtained by treating with a fluorochrome or the fluorescent dye quinacrine. They can be identified by a yellow fluorescence of different intensity. • Most parts of the stained DNA are heterochromatin. • A-T regions are seen more in heterochromatin than in euchromatin. Therefore, by this banding method heterochromatin regions are labeled preferentially. • The characters of the banding regions and the specificity of the fluorochrome are not exclusively dependent on their affinity to regions rich in A- T, but it depends on the distribution of A- T and its association with other molecules such as histone proteins. Advantages:  It is a simple and versatile technique,  It is used where G – band is not acceptable. It is used as a method of identifying chromosomes in combination with other procedure.  Study of heteromorphism  Study of human Y chromosome Disadvantages:  Generally associated with any fluorescence technique: impermanence of the preparations, the tendency to fade during examination.

2. G-banding: • Giemsa has become the most commonly used stain in cytogenetic analysis. Staining a metaphase chromosome with a Giemsa stain is referred to as G-banding. This technique is not a fluorochrome -based pretreatment. • • • It is well suited to animal cells. During mitosis, the 23 pairs of human chromosomes condense and are visible with a light microscope. A karyotype analysis usually involves blocking cells in mitosis and staining the condensed chromosomes with Giemsa dye. The dye stains regions of chromosomes that are rich in the base pairs Adenine (A) and Thymine (T) producing a dark band. The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.

the chromosomes with either salt or a proteolytic (protein-digesting) enzyme. "GTG banding" refers to the process in which G-banding is preceded by treating chromosomes with trypsin. A common misconception is that bands represent single genes, but in fact the thinnest bands contain over a million base pairs and potentially hundreds of genes. For example, the size of one small band is about equal to the entire genetic information for one bacterium. • Standard G-band staining techniques allow between 400 and 600 bands to be seen on metaphase chromosomes.

G-banded metaphase from a normal female.

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With high-resolution G-banding techniques, as many as two thousand different bands have been catalogued on the twenty-four human chromosomes.

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Jorge Yunis introduced a technique to synchronize cells so they are held at the same stage in the cell cycle. Cells are synchronized by making them deficient in folate, thereby inhibiting DNA synthesis. By rescuing the cells with thymidine, DNA synthesis is initiated and the timing of the prophase and prometaphase stages of the cell cycle can be predicted. Yunis's technique allows more bands to be resolved, as chromosomes produced from either prophase or prometaphase are less condensed and are thus longer than metaphase chromosomes.

3. C-banding: • • • The name C-banding originated from centromeric or constitutive heterochromatin. Cbanding stains areas of heterochromatin, which is tightly packed and repetitive DNA. The centromere appears as a stained band compared to other regions. The technique involves a pretreatment with alkali before staining. The alkaline pretreatment leads to the complete depurination of the DNA. The remaining DNA is again renatured and stained with Giemsa solution consisting of methylene azure, methylene violet, methylene blue, and eosin. • In this staining the heterochromatin take a lot of stain but the rest of the chromosomes stain only a little. Applications:  This banding technique is well suited for the characterization of plant chromosomes.

7  C banding is valuable for the identification of chromosome particularly in insects of plants  Useful in the identification of meiotic chromosomes even in the species such as mammals which shows good G banding pattern on mitotic chromosome.  C banding is valuable to identify bivalents at diakinesis using both centromere positions.  C bands used for paternity testing and gene mapping. 4. R-banding: • This is known as a reverse banding technique. This technique results in the staining of areas rich in G-C that is typical for euchromatins. • R-banding involves pretreating cells with a hot salt solution that denatures DNA that is rich in adenine and thymine. The chromosomes are then stained with Giemsa. • Advantage - R-banding is helpful for analyzing the structure of chromosome ends, since these areas usually stain light with G-banding. • G-, Q-, and R-bandings are not observed with plant chromosomes.

5. Hy-banding: • • • This is a common technique used with plant cells. The technique involves a pretreatment of the cells in which the cells are warmed in the presence of HCl and then stained with acetocarmine. The pattern of Hy-band is different from that of C-bands. The binding of histone protein to DNA and its complete extraction has an impact on the binding ability of acetocarmine and formation of bands.

6. NOR-staining: (Silver Nucleolar Organizing Region Staining)

8 • NOR is an abbreviation for "nucleolar organizing region," refers to a silver staining method that identifies genes for ribosomal RNA that were active in a previous cell cycle. • Chromosomes are treated with silver nitrate solution which binds to the Nucleolar Organizing Regions (NOR), i.e., the secondary constrictions (stalks) of acrocentric chromosomes.

7. DAPI/Distamycin A Staining • The DAPI/distamycin- A fluorescent staining technique was first described by Schweizer, Ambros, and Andrle as a method for labeling a specific subset of C bands. (Gustashaw, 1991). • The combination of the fluorescent dye, DAPI (4, 6-Diamidino-2-Phenylindole) with a non-fluorescent counterstain, such as Distamycin A, will also stain DNA that is rich in adenine and thymine. • It will particularly highlight regions that are on the Y chromosome, on chromosomes 9 and 16, and on the proximal short arms of the chromosome 15 homologues, or pair. • The DAPI/Distamycin A staining technique is useful in identifying pericentromeric breakpoints in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques.

9. T-Banding: • • T-banding is used to stain the telomeric regions of chromosomes for cytogenetic analysis. Telomeric (or terminal) banding was first reported by Dutrillaux, who used two types of controlled thermal denaturation followed by staining with either Giemsa or acridine orange.

9 • The T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric. (Gustashaw, 1991).